SummaryObjective  It has been reported that macroprolactin is a complex of PRL and an immunoglobulin G (IgG). This study further characterizes macroprolactin and evaluates for other markers of autoimmunity using a cohort of macroprolactinaemic sera.Patients and normal subjects  Following treatment of hyperprolactinaemic sera (n = 58) with polyethylene glycol (PEG), PRL values fell from 524–13 546 mU/l (Range) to 452–8455 mU/l, while in macroprolactinaemic sera (n = 41), PRL concentration fell from 525–5747 to 98–378 mU/l (PEG treated normoprolactinaemic reference range, 68–230 mU/l in males, 70–390 mU/l in females).Design  PRL was measured in sera prior to and following gel filtration chromatography, ultrafiltration, treatment with protein A‐sepharose, protein G‐sepharose, antihuman IgG‐agarose and sodium thiocyanate (NaSCN). The binding of radio‐labelled PRL in macroprolactinaemic sera was also measured. Sera were assayed for antithyroid and antinuclear antibodies. C‐reactive protein (CRP) and CD5 positive B cells were also measured. Comparisons were made between values obtained in normal, hyperprolactinaemic and macroprolactinaemic sera.Results  Macroprolactinaemic sera indicated the presence of an IgG molecule and/or IgG fragments with one or more molecules of PRL. In 97% of the sera macroprolactin had a molecular weight of 204 kDa. Treatment of macroprolactinaemic sera with NaSCN caused dissociation of macroprolactin, releasing monomeric PRL. Macroprolactinaemic sera did not yield evidence of an increase in markers of autoimmunity when compared with hyperprolactinaemic or normal sera.Conclusions  Comprehensive analysis of macroprolactin confirmed its composition as an IgG molecule or fragment with a PRL molecule. The occurrence of macroprolactin does not appear to be associated with autoimmunity.